Effects And Mechanisms Of SEMA3A In Secondary Brain Damage Following Traumatic Brain Injury

Objective: Traumatic brain injury(TBI)is a disease that leads to high disability and mortality around the world.Nowadays,there are still lacking an effective therapy and mechanism of the secondary injury post-TBI.SEMA3 A has been reported to affect vascular permeability,but its effect in TBI is still unknown.We focus on the effect of SEMA3 A on BBB damage and neuron apoptosis after TBI,and its regulation,and try to find a new treatment target and clinical therapy for TBI.Method: We used a mouse TBI model with CCI and C57/BL6 J mice.Collected the brain tissue on 1 3,7,14 day post-TBI and sham group.Tested the expression of SEMA3 A and its associated receptor(Nrp-1 and Plexin-A1)with western blot.We also tested the expression area on brain tissue through immune fluroscence.We transfected si RNA-SEMA3 A or recombinant SEMA3 A to alter the expression of SEMA3 A in mice brain tissue.We used m NSS Score and beam walking test to test the neurological outcomes post-TBI.Furthermore,we used Evans blue assay to test the interruption of BBB and evaluated the brain edema with water content assay.Finally,we tested the expression of BBB associated junction protein with western blot.We used b End.3 to create BBB model and carried out OGD model to imitate TBI in vitro.First,we tested the expression of SEMA3 A and its associated receptor after OGD with western blot.And then,we tested the BBB leakage with dextran transport assay.The morphology of endothelial barrier was observed by HPICM scanning.Finally,we tested the expression of BBB associated junction protein with western blot.We carried out q RT-PCR to test the expression of miRNA-30b-5p.Next,we used agomir and antagomir to change the expression level of miRNA-30b-5p in vivo.We also used mimic and inhibitor to alter the expression of miRNA-30b-5p in vitro.The expression of SEMA3 A was tested by western blot.The target relationship was confirmed through luciferase assay.Finally,the neuroprotective function of mi R-30b-5p is measured by brain water content,m NSSs and beam-walking test.Finally,we use TUNEL to detect the neuron apoptosis after TBI.Detect expression level of cleaved caspase-9 and-3 through western blot.Next,we used agomir and detect expression level of cleaved caspase-9 and-3 through western blot.Result:1.(1)Expression of SEMA3 A and related receptors post-TBI was increased post-TBI.(2)SEMA3A was mainly secreted and concentrated at lesion boundary after TBI.2.(1)Downregulation of SEMA3 A decreased m NSS scores and improve motor function post-injury.(2)SEMA3A can increase BBB leakage and brain edema,and can be decreased by blocking SEMA3 A.(3)Dcreasing SEMA3 A can inhibit VE-cadherin serine phosphorylation post-TBI.3.(1)The expression of SEMA3 A and related receptors increased in OGD group.(2)Dextran leakage was increased in the OGD group and the SEMA3 A group.This amount decreased when SEMA3 A was blocked.(4)The HPICM results illustrated that SEMA3 A can efficiently increase defects of the BBB.Observations also indicated that the destruction of the barrier that is impaired by downregulation of SEMA3 A.(5)SEMA3A contributes to VE-cadherin serine phosphorylation post-OGD.4.(1)The q RT-PCR results indicated that the expression level of mi R-30b-5p in vitro and in vivo experiment was decreased and upregulation of mi R-30b-5p decreased SEMA3 A levels.(2)Luciferase data suggested that mi R-30b-5p could directly target Sema3 a and downregulate its expression by binding to the 3’UTR sites.(4)Upregulation of mi R-30b-5p could efficiently decrease negative effect of SEMA3 A post-TBI.5.(1)Decreasing SEMA3 A can improve the neuron apoptosis post-TBI.(2)Decreasing SEMA3 A can decrease the expression of cleaved caspase-9 and-3,and can be improved by mi R-30b-5p.Conclusion:1.SEMA3 A and relative receptors increased after TBI and concentrated on leision boundary.2.Decreasing SEMA3 A can improve BBB damage,brain edema and neurological dysfunction post-TBI.3.OGD can increase the expression of SEMA3 A and its relative receptors in b End.3.Decreasing SEMA3 A can improve endothelial cell barrier leakage.4.miRNA-30b-5p increased after injury in vivo and in vitro.miRNA-30b-5p can decrease the expression of SEMA3 A though binding to3’UTR region of sema3 a.5.SEMA3 A contributed to neuron apoptosis post-TBI through caspase-9/caspase-3 pathway and can be improved by miRNA-30b-5p.